Conjugation to 4-aminoquinoline improves the anti-trypanosomal activity of Deferiprone-type iron chelators

Article abstract of DOI:10.1016/j.bmc.2012.11.009

Iron is an essential growth component in all living organisms and plays a central role in numerous biochemical processes due to its redox potential and high affinity for oxygen. The use of iron chelators has been suggested as a novel therapeutic approach

Full text of DOI:10.1016/j.bmc.2012.11.009

Bioorganic & Medicinal Chemistry 21 (2013) 805–813
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Conjugation to 4-aminoquinoline improves the anti-trypanosomal activity
of Deferiprone-type iron chelators
Sebastian S. Gehrke a,b,c, Erika G. Pinto d,e, Dietmar Steverding c, Karin Pleban a, Andre G. Tempone d,
Robert C. Hider a, Gerd K. Wagner a,f,
⇑
a Institute of Pharmaceutical Science, School of Biomedical Sciences, King’s College London, SE19NH, UK
b School of Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK
c BioMedical Research Centre, Norwich Medical School, University of East Anglia, Norwich NR4 7TJ, UK
d Department of Parasitology, Instituto Adolfo Lutz, São Paulo, Brazil
e Instituto de Medicina Tropical, Universidade de São Paulo, São Paulo, Brazil
f Department of Chemistry, School of Biomedical Sciences, King’s College London, SE19NH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Iron is an essential growth component in all living organisms and plays a central role in numerous bio-
chemical processes due to its redox potential and high affinity for oxygen. The use of iron chelators has
been suggested as a novel therapeutic approach towards parasitic infections, such as malaria, sleeping
sickness and leishmaniasis. Known iron chelating agents such as Deferoxamine and the 3-hydroxypyri-
din-4-one (HPO) Deferiprone possess anti-parasitic activity but suffer from mammalian toxicity, rela-
tively modest potency, and/or poor oral availability. In this study, we have developed novel derivatives
of Deferiprone with increased anti-parasitic activity and reduced cytotoxicity against human cell lines.
Of particular interest are several new derivatives in which the HPO scaffold has been conjugated, via a
linker, to the 4-aminoquinoline ring system present in the known anti-malaria drug Chloroquine. We
report the inhibitory activity of these novel analogues against four parasitic protozoa, Trypanosoma bru-
cei, Trypanosoma cruzi, Leishmania infantum and Plasmodium falciparum, and, for direct comparison,
against human cells lines. We also present data, which support the hypothesis that iron starvation is
the major cause of growth inhibition of these new Deferiprone–Chloroquine conjugates in T. brucei.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 6 October 2012
Revised 8 November 2012
Accepted 9 November 2012
Available online 27 November 2012
Keywords:
Iron chelator
Anti-parasitic
Trypanosoma
Hybrid drugs
Deferiprone
1. Introduction
deaths per year due to cardiac disease.4 Cases of Visceral
Leishmaniasis (VL) have been reported in 98 countries, with an
Neglected tropical diseases such as malaria, sleeping sickness
and leishmaniasis continue to pose a very considerable health
threat to significant parts of the population, particularly in the
developing world.1 The causative agents of these diseases are dif-
ferent parasitic protozoa, namely Plasmodium falciparum (malaria),
Trypanosoma brucei (human African sleeping sickness), Trypano-
soma cruzi (Chagas disease) and Leishmania sp. (leishmaniasis). In
2008, the WHO recorded nearly 1 million deaths caused by malar-
ia, mostly among African children.2 Cases of African sleeping sick-
ness, also known as Human African Trypanosomiasis (HAT), have
been reported in 36 sub-Saharan countries, and despite significant
progress in the control and treatment of HAT over the past decade,
the estimated incidence of HAT still reaches 10,000 cases per
annum.3 Chagas disease is a major cause of morbidity and mortal-
ity in many regions of South America, with an estimated preva-
lence of 16–18 million people infected and approximately 45,000
estimated prevalence of 12 million cases worldwide and an
incidence of 500,000 new cases per year.5 If untreated, all of these
diseases, especially HAT and VL, can be fatal.
Current treatment options for these parasitic infections focus
mainly on chemotherapy. However, existing therapies often rely
on outdated and toxic drugs that were identified decades ago, such
as the arsenical melarsoprol for HAT6 and pentavalent antimonials
for VL.7 In the case of Chagas disease, benznidazole and nifurtimox
are the only available drugs for the treatment of acute infections,
but are ineffective and too toxic against the chronic stage of the
disease.8 In addition, the clinical use of both drugs can be compro-
mised by significant side effects such as peripheral polyneuropa-
thy, depression of bone marrow, and allergic dermopathy.9 These
limitations of the few existing treatments for parasitic infections,
together with growing resistance development, have created an ur-
gent medical need for the development of new anti-parasitic
agents.1 The identification of such agents, ideally with oral avail-
ability and broad-spectrum activity against several of these para-
sites, is therefore a highly topical area of research.10–13
⇑
Corresponding author.
E-mail address: gerd.wagner@kcl.ac.uk (G.K. Wagner).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.009

806
S. S. Gehrke et al. / Bioorg. Med. Chem. 21 (2013) 805–813
Table 1
Activity of reference compounds Chloroquine, Desferoxamine (DFO) and Deferiprone, and of Deferiprone–CQ hybrid 2c, against different parasites and mammalian cell lines
O
O
OH
N
HN
OH
N
CH3
2c
N
CH3
CH3
4
HN
N
Cl
N
Chloroquine
Deferiprone (1a)
Cl
O
OH
N
OH
N
O
H
H2N
N
CH3
N
N
H
O
OH
O
O
Desferoxamine
GI50 (lM)
CQ
DFO
Deferiprone (1a)
Hybrid (2c)
P. falciparum
T. brucei
T. cruzi
L. infantum
HL60
0.0025a
15.6
22.2
3.7
28
67b
>100
2.4
3.8
6.5
27.0
50.3
38.2
2.9c
n.a.d
150e
18.4c
33.1
>100
75e, >100f
>100
128
K562
n.a.d
n.a.d
a
b
c
d
e
f
From Ref. 13.
From Ref. 25.
From Ref. 18.
Data not available.
From Ref. 36.
This study.
Iron has a central role in numerous biochemical processes due
to its redox potential and high affinity for oxygen.14 In protozoa,
as well as fungi and bacteria, iron is an essential growth factor,15
and iron chelation has been suggested as a potential new strategy
to combat parasitic infections.16 It has previously been shown that
the iron chelator desferoxamine (DFO, Table 1) compromises the
activity of Fe(III)-containing enzymes in parasitic protozoa such
as ribonucleotide reductase, which is directly involved in DNA syn-
thesis, by chelating cellular iron.17–20 Consequently, iron chelators
interfere with the growth of protozoan parasites, and DFO has been
reported to successfully clear human hosts from P. falciparum in
over 90% of those treated,21 and to also possess cytotoxicity against
T. brucei.18,19 While these results illustrate the potential of DFO and
related iron chelators for anti-parasitic applications, the clinical
use of DFO itself is compromised by its poor oral availability.21,22
In contrast to DFO, iron chelators of the 3-hydroxypyridin-4-
one (HPO) class such as the marketed drug Deferiprone (1a, Ta-
ble 1) are orally active, which is partly due to their relatively
low-molecular weight compared to DFO. Despite a molecular
weight of <140, 1a and related HPOs are effective iron chelators
with a high selectivity for Fe(III), due to the formation of 3:1 com-
plexes with the metal (Fig. 1). Deferiprone itself, which is used clin-
ically for the treatment of iron overload,23 has also been assessed
for its anti-malarial potential. Although the drug has moderate
in vitro activity against P. falciparum (Table 1), its in vivo anti-
malarial activity, at dosages that are effective for treating iron
overload, is poor.24 Structural modifications in position N1, includ-
ing the introduction of acidic functionalities, did not significantly
improve the in vitro activity of 1a.21,25 In addition, 1a as well as
these N1-modified derivatives had only moderate selectivity for
P. falciparum and also showed significant cytotoxicity against
mammalian cells.25,26
H3C
N
O
O
O
O
O
Fe3+
N
CH3
O
CH3
N
Figure 1. Deferiprone (1a) in complex with Fe(III).
conjugated, via a linker at N1, to the 4-aminoquinoline ring system
present in the known anti-malarial drug Chloroquine. Previously,
such a conjugation strategy with Chloroquine has been used suc-
cessfully with other bioactive moieties.27–31 Herein we present
the results from the application of this approach to iron chelators
of the HPO class. We have synthesized a series of new Deferi-
prone–Chloroquine hybrids and investigated their activity against
P. falciparum and, in two cases, also against three other protozoal
parasites (T. brucei, T. cruzi and Leishmania infantum). Although con-
jugation to Chloroquine resulted in improved anti-malarial activity
relative to the parent 1a, the new hybrids were less active than
Chloroquine alone. Intriguingly, however, one of the new Deferi-
prone–Chloroquine hybrids showed increased activity against both
trypanosome species (T. brucei and T. cruzi) compared to both 1a and
Chloroquine. Previously, 1a had never been evaluated against
Trypanosoma, and these are the first examples for the anti-trypanos-
omal activity of this class of iron chelator. Additional mode-of-
action experiments with T. brucei strongly indicate that iron
deprivation contributes significantly to the anti-trypanosomal
activity of the new Deferiprone–Chloroquine conjugates. Taken
together, our results suggest that the conjugation of HPO iron
In order to improve the anti-parasitic activity of 1a, we have de-
signed novel derivatives of 1a, in which the HPO scaffold has been

S. S. Gehrke et al. / Bioorg. Med. Chem. 21 (2013) 805–813
807
NH2
N
O
O
Cl
N
OH
OBn
CH3
(vi)
Cl
Cl
O
CH3
O
4
5
7
maltol
(vii)
(i)
(ii)
O
N
NH2
n
OR
O
HN
N
CH3
OBn
CH3
(iii)
(viii)
n
HN
Strategy A
(6a-c)
N
Strategy B
Cl
(6c & d)
4
3a (n = 2)
3b (n = 3)
3c (n = 4)
H2N
R1
N
R2
8
(iv) or (v)
6
2
protected species deprotected species deprotection
R1 R2
n
(R = Bn)
(R = H)
conditions
6a
6b
6c
6c
2a
2b
2c
2d
(iv)
(iv)
(iv)
(v)
Cl
Cl
Cl
H
H
H
H
H
2
3
4
4
4
CH3
6d
2e
(iv)
H
Scheme 1. Synthesis of HPOs 2a–e conjugated to Chloroquine and Chloroquine derivatives. Reagents and conditions: (i) 2-(4-bromobutyl)isoindoline-1,3-dione, NaHCO3,
DMF or DMSO or MeCN or EtOH; (ii) 3a: ethylenediamine (9 equiv), reflux, 12 h; 3b: 1,3-diaminopropane (7 equiv), reflux, 12 h; 3c: 1,4-diaminobutane (5 equiv), 160–180 °C,
18 h; (iii) 7, 2 N NaOH, pH 13, aqueous EtOH (25%), reflux, 12 h; (iv) 6 N HCl, reflux, 2 h; (v) 5% Pd/C, H2, ethanol, aq HCl; (vi) NaOH (1.1 equiv), BnBr (1.1 equiv), aq MeOH
(50%), reflux, 6 h; (vii) 1,4-diaminobutane (1 equiv), 10 N NaOH, pH 13, reflux, 18 h; (viii) 6c: 4,7-dichloroquinoline (1 equiv), or 6d: 4-chloroquinaldine (1 equiv), DMSO,
100–110 °C, 12 h.
chelators to a second bioactive moiety is a promising strategy
against Trypanosoma parasites.
this reactivity problem, we switched to a SnAr strategy, as also de-
scribed recently by Pérez et al.28 Thus, 4,7-dichloroquinoline 5 was
reacted in neat solution of different diaminoalkanes to provide the
desired amines 3a–c in moderate yields (48–66%). Conveniently,
3a–c were afforded as white crystals after re-crystallization from
ethanol/ether. In the next step, the primary amines 3a–c were re-
acted with benzyl-protected maltol 7 in aqueous ethanol and in
the presence of catalytic amounts of sodium hydroxide to give the
double Michael addition products 6a–c. Interestingly, we found that
the base-catalyzed Michael addition reaction is pH sensitive. The
best yields were achieved at pH 13. Finally, removal of the benzyl
protecting group under acidic conditions gave the target HPOs 2a–c.
HPOs 2d–e were obtained via synthetic strategy B, in which the
N-substituted HPO core was prepared by a double Michael addi-
tion of 1,4-diaminobutane to 7 (Scheme 1). The resulting primary
amine 8 was then used in an SN reaction with, respectively, 4,7-
dichloroquinoline or 4-chloroquinaldine, to provide the benzyl-
protected HPOs 6c and 6d. Removal of the benzyl protecting group
in 6c under acidic conditions provided an alternative synthetic en-
try to HPO 2c, while deprotection of 6c by catalytic dehydrogena-
tion led to concomitant dehalogenation in position 7, thus
affording HPO 2d. Analogously, deprotection of 6d by catalytic
hydrogenation gave target HPO 2e.
2. Chemistry
The new Deferiprone derivatives 2a–e, which are conjugated via
an alkyl linker to various 4-aminoquinolines, were prepared in anal-
ogy to our previously reported syntheses for simple N1-substituted
HPOs (Scheme 1).32 The central step in this approach is the installa-
tion of the N1-substituent via the double Michael addition of a suit-
able primary amine to the benzyl-protected maltol derivative 7. In
order to prepare the HPO derivatives 2a–e, we employed two differ-
ent variations of this general strategy (Scheme 1). For the prepara-
tion of HPOs 2a–c, we followed strategy A, which is characterized
by the installation of the linker at the Chloroquine fragment prior
to the double Michael addition. For this strategy, the primary amines
3a–c were required as intermediates. In initial trials, all attempts to
alkylate aminoquinoline
4 by reaction with N-(4-bromobu-
tyl)phthalimide were unsuccessful, probably due to the limited
nucleophilicity of the aromatic amino group. In order to overcome
O
N
O
N
OBn
CH3
OH
Finally, we also prepared derivative 10 in which the linker con-
tains an amide functionality (Scheme 2). For the preparation of 10,
7 was reacted with c-aminobutyric acid (GABA) to afford the free
(i)
(ii),(iii)
CH3
7
carboxylic acid 9. Standard coupling conditions with DCC, DMAP
and 4-aminoquinaldine gave the desired amide and, after removal
of the benzyl protecting group via catalytic hydrogenation, the tar-
get HPO 10.
2
2
HO
O
HN
N
O
9
10
CH3
3. Biological results
Scheme 2. Synthesis of HPO 10 containing an amide group in the linker. Reagents
and conditions: (i) GABA (1.2 equiv), aq EtOH (50%), 10 N NaOH, pH 13, reflux, 18 h;
(ii) 4-aminoquinaldine (1 equiv), DCC (1.2 equiv), DMF, 70 °C, 12 h; (iii) Pd/C, H2,
ethanol, pH 1 (HCl).
We have previously shown that Deferiprone (1a) has modest
activity against P. falciparum (Table 1).25 Conjugation of the HPO

808
S. S. Gehrke et al. / Bioorg. Med. Chem. 21 (2013) 805–813
Table 2
mal activity of 1a and its derivatives 2c and 2d, we used
established viability assays for these parasites.33–35 While the par-
ent compound 1a (Deferiprone) had moderate activity against Plas-
modium, it was inactive against T. brucei and T. cruzi at
Anti-plasmodium activity of HPOs 2a–e and 10
O
O
N
OH
OH
concentrations up to 100 lM (Table 1). The Deferiprone conjugates
N
CH3
CH3
2c and 2d, on the other hand, showed activity against T. brucei at
low micromolar concentrations, similar to their activity against P.
falciparum (Table 3). Compound 2c, but not 2d, was also active
against T. cruzi. This anti-trypanosomal activity cannot be attrib-
uted exclusively to the presence of the Chloroquine fragment, as
both CQ itself and the Chloroquine-based synthetic precursor 3c,
which corresponds to the Chloroquine-conjugate 2c, but without
the HPO fragment, were four- to sixfold less active against both
trypanosome species than 2c.
n
HN
N
2
HN
O
R1
R2
10
2a-e
N
CH3
M)
Compd
n
R1
R2
GI50
(
l
SIb
P. falciparuma
K562
Chloroquine-conjugate 2c, but not its close structural analogue
2d, also displayed activity against L. infantum promastigotes in our
anti-leishmanial assay (Table 3).34 Against this parasite, 2c was at
least four times more active than its parent Deferiprone 1a, which
did not show any activity in our own experiments at concentra-
2a
2c
2d
2e
10
2
4
4
4
Cl
Cl
H
H
n/a
H
H
H
CH3
n/a
7.0
2.4
1.6
9.0
6.0
182.2
38.2
27.1
204.2
127.7
26
16
17
23
21
n/a
tions up to 100 lM (Table 1), even though moderate activity
a
Strain ITG2G1.
Selectivity index.
against L. infantum has previously been reported for 1a.36 Leish-
mania promastigotes were approximately fourfold more resistant
to 2c than T. cruzi trypomastigotes. It has previously been observed
that Leishmania parasites are less susceptible to drugs than T. cru-
zi.35 The different bioactivities of the new HPO-conjugate 2c
against these two parasites are therefore not entirely unexpected.
Compound 2c was also tested against the intracellular amastigote
forms of L. infantum, but showed no activity at the highest concen-
b
Table 3
Anti-trypanosomal and anti-leishmanial activity of HPOs 1a–d, 2c, 2d and 3c
O
OH
NH2
tration that was tested (60 lM). This may be due to the different
O
metabolism of amastigotes, which live in the unusual intracellular
milieu of macrophages, and/or the limited uptake of 2c by macro-
phages. In this context it is interesting to note that the substitution
of Cl (2c) with H (2d) abolished the toxicity to mammalian cells,
but also reduced anti-leishmanial activity.
4
N
CH3
HN
OH
4
HN
N
N
R
CH3
Cl
N
X
1a-d
3c (X = Cl)
Taken together, these results suggest that conjugation of 1a to a
Chloroquine-like fragment leads to synergistic, anti-parasitic ef-
fects, which are particularly pronounced against trypanosomes,
but more modest against Leishmania. To understand the role of
the Chloroquine fragment for the anti-parasitic activity of the
new HPO–Chloroquine conjugates, we also investigated the
activity of selected Deferiprone derivatives with simple alkyl sub-
stituents in position N1 against T. brucei, T. cruzi and L. infantum.
Interestingly, the octyl- and decyl-substituted derivatives 1b and
1c showed potent activity in the low micromolar range against
all three parasites (Table 3). However, both iron chelators were
also cytotoxic towards HL-60 cells at only slightly higher concen-
trations, resulting in a relatively modest selectivity index (Table 4).
Previously, partition coefficients (logP values) close to 1 have been
2c (X = Cl)
2d (X = H)
Compd
R
GI50
(l
M)
T. brucei
>100
T. cruzi
>100
3.0 0.3
0.8 0.1
>100
6.5 0.1
>100
46.3 13.4
L. infantuma
HL60
>100
11.2 0.4
6.3 0.2
111
50.3 1.1
>100
1a
1b
1c
1d
2c
2d
3c
CH3
C8H17
>100
3.6 0.2
1.4 0.05
>100
3.8 1.0
7.6 0.4
15.8 1.3
3.2 0.4
0.7 0.03
>100
27.1 7.0
>100
C
10H21
C2H4NH2
n.a.
n.a.
2
n.a.
42.4 4.8
>100
a
Promastigote form.
scaffold in 1a via a linker to different 4-aminoquinolines, the phar-
macophore of the known anti-malarial Chloroquine (CQ) signifi-
cantly improved anti-plasmodial activity (Table 2). All of the new
HPO-conjugates 2a–e and 10 showed enhanced anti-plasmodial
activities compared to the parent 1a, as well as a favorable cytotox-
icity profile, with 16- to 26-fold selectivity for P. falciparum over a
mammalian cell line. However, 2a–e and 10 were less active than
CQ alone. While low micromolar activity against P. falciparum was
observed with all analogues in this series, irrespective of the nature
or length of the linker, the best results were achieved with ana-
logues 2c and 2d, both in terms of anti-plasmodium activity and
parasite selectivity. We therefore selected these two conjugates
for further studies with other parasites.
Table 4
Selectivity indices and logP values of HPOs 1a–c, 2c and 2d
Code
R
X
SIa
LogPb
T. brucei
T. cruzi
L. infantum
1a
1b
1c
2c
2d
CQ
CH3
C8H17
n/ac
n/ac
n/ac
Cl
n.d.d
3
5
13
>13
8
n.d.d
n.d.d
4
9
0.40
3.56
4.45
1.40
1.25
n.d.d
4
8
8
C
10H21
n/ac
n/ac
n/ac
2
H
n.d.d
6
n.d.d
35
n/ac
a
Selectivity index versus HL60 cells.
Calculated clogP values for 1a–1c, experimentally determined logP values for
While Deferiprone (1a) and some N1-substituted derivatives
have previously been tested against P. falciparum and Plasmodium
berghei,21,25,26 the activity of this class of iron chelators against Try-
panosoma has never been evaluated. To assess the anti-trypanoso-
b
2c and 2d, see Supplementary data for details.
c
Not applicable.
Not determined.
d

S. S. Gehrke et al. / Bioorg. Med. Chem. 21 (2013) 805–813
809
suggested as favorable for both inhibition of iron release and mem-
brane permeability,26 while higher logP values have been associ-
ated with increased toxicity of Deferiprone derivatives.26 This
correlation between lipophilicity and toxicity appears to hold for
the new derivatives 1b and 1c, which both have clogP values
greater than 3.5 (Table 4). Experimentally determined logP values
for Chloroquine-conjugates 2c and 2d, on the other hand, were sig-
nificantly lower, and both 2c and 2d had a more favorable selectiv-
ity profile than either 1b, 1c or Chloroquine alone, at least against
T. brucei (Table 4). A possible explanation for these results is that
Deferiprone analogues with a simple alkyl substituent in position
N1 (e.g., 1b, 1c) may act non-specifically on iron depletion within
the cellular compartment of both parasite and mammalian cells,
resulting in their general cytotoxicity. In contrast, Chloroquine-
conjugates 2c and 2d may have a certain preference for parasite
cells, at least in the case of T. brucei. An alternative explanation
for the non-specific cytotoxicity of 1b and 1c may be that mole-
cules with a long N-alkyl substituent attached to a hydrophilic
fragment can induce membrane disruption.37
To further investigate the anti-parasitic activity of the hybrid
molecules, we next carried out mode-of-action studies. First, we
determined important physicochemical parameters for the new
HPO–Chloroquine conjugates, namely their pKa values and abso-
lute stability constant for Fe(III), pFe3+. The HPO–Chloroquine con-
jugates are characterized by three possible protonation states pKa1
(protonation of the 4-oxo group), pKa2 (protonation of the quino-
line ring nitrogen) and pKa3 (dissociation of the 3-OH group; Sup-
plementary data). For compounds 2a–2e, pKa2 values range from
7.5 to 9.6, indicating that a significant proportion of each com-
pound is protonated at pH 7.4 (Supplementary data). Conse-
quently, the neutral fraction of each HPO–Chloroquine conjugate
is relatively small, and only a minor percentage would be expected
to readily cross biological membranes through passive diffusion. 2e
with the highest pKa2 of all compounds has the lowest distribution
coefficient D7.4, whereas 2c with an pKa2 of 8.2 has the highest D7.4
(Supplementary data). This indicates that in this series, a pKa2 of
ꢀ8 leads to the best distribution profile, in keeping with the supe-
rior bioactivity of 2c and 2d against P. falciparum.
media. Using a spectrophotometric titration method, we deter-
mined the pFe3+ value of 2e as 21.1 at pH 7.45. Interestingly, this
value falls between the pFe3+ values of 1a (19.3) and DFO (26.6).
This result therefore suggests that conjugation to CQ does not
interfere with the iron chelation capacity of HPOs, and that the
new HPO–Chloroquine conjugates remain potent iron chelators.
Finally, we investigated the relevance of iron chelation for the
anti-trypanosomal activity of the HPO–Chloroquine conjugates in
live trypanosomes. For these mode-of-action experiments we fo-
cused on T. brucei, as relatively straightforward assays exist to
investigate the effect of iron depletion with bloodstream forms of
this parasite. It has previously been shown that under conditions
of iron deprivation, T. brucei upregulates the expression of their
transferrin receptor (TbTfR).38 TbTfR expression therefore is an
indicator of the parasite’s iron status, and we studied the upregu-
lation of TbTfR by the parasite in the presence of 2d. As a measure
of TbTfR upregulation, we quantified the uptake of fluorescently la-
beled transferrin by T. brucei using FACS analysis. Under these con-
ditions, the parasite showed an increased uptake of transferrin by a
factor of 1.6 in the presence of 2d, compared to control cells incu-
bated with DMSO (Fig. 2). Transferrin uptake was also increased,
by a factor of 2.0, by the known iron chelator DFO. These results
suggest that the capacity of 2d to act as an iron chelator under cel-
lular conditions is responsible, at least in part, for its anti-trypan-
osomal activity. This interpretation is further confirmed by the
observation that pre-incubation of 2d with FeCl3, and thus satura-
tion of the iron chelator with Fe(III), results in a complete loss of
activity against T. brucei in our cytotoxicity assay. It should be
noted that the iron-saturated compound 2d is not charged but
neutral (Fig. 1) and therefore able to penetrate the parasite plasma
membrane and diffuse into the cytosol. However, the excess con-
centration of free Fe(III) ions in solution most likely scavenges all
of the iron chelator, leaving the parasite with enough iron (e.g.,
from transferrin) to maintain viability.
4. Conclusion
We describe herein the anti-parasitic activity of several deriva-
tives of the known iron chelator Deferiprone (1a). Derivatives of
HPO 1a with simple alkyl substituents in position N1 are generally
cytotoxic both against parasites and mammalian cells. In contrast,
a new class of N1-substituted HPO derivatives conjugated to the
known anti-malarial Chloroquine showed preferential activity
against parasitic cells, in particular in the case of P. falciparum
and T. brucei. The new HPO derivatives retain their potent iron che-
lating capacity, which very likely forms the basis for their anti-
parasitic activity. Importantly, the activity of the most potent con-
jugate 2c against T. brucei and T. cruzi is clearly improved relative
to both parent compounds. Thus, the anti-trypanosomal activity
of 2c was increased >25-fold against T. brucei and >15-fold against
T. cruzi compared to Deferiprone, and about fourfold against both
species compared to Chloroquine.
These results represent a promising starting point to revisit the
application of iron chelators, in particular of the HPO class, as
anti-parasitic agents. Our findings suggest that, at least against try-
panosomes, our conjugation strategy leads to synergistic effects
between the HPO and Chloroquine fragments, while retaining rea-
sonable selectivity indices. The good anti-trypanosomal activity is
particularly remarkable, as neither Deferiprone, nor Chloroquine
alone, are potent anti-trypanosomal agents. Results from pKa stud-
ies indicate that the neutral, membrane-permeant fraction of even
the best HPO–Chloroquine conjugates in this series is relatively
small. Modification of their acid/based properties may therefore
represent a promising strategy for further optimisation of the
anti-trypanosomal activity of this class of iron chelators.
To assess the efficacy of HPO–Chloroquine conjugates as iron
chelators we next determined pFe3+ over a pH range of 2–11. For
these experiments, 2e was selected as the best suited HPO–Chloro-
quine conjugate due to its relatively good solubility in aqueous
4000
3000
2000
1000
0
DMSO
CP421
DFO
Figure 2. Accumulation of fluorescein-labeled transferrin in bloodstream forms of
T. brucei upon incubation with 2d (CP421) and DFO. Conditions: Trypanosomes
(5 ꢁ 105/mL) were incubated with 50
lM 2d (CP421), 25 lM DFO or DMSO
(control) in Baltz medium supplemented with 16.7% heat-inactivated FBS. After
24 h incubation, trypanosomes were harvested and washed three times with Baltz
medium supplemented with 2% BSA. Then, the cells were incubated with 50 lg/mL
fluorescein-labeled transferrin in Baltz medium/2% BSA for 2 h. After washing twice
with PBS/1% glucose, cells were fixed with 2% formaldehyde/0.05% glutaraldehyde,
and analyzed by flow cytometry using a DB Accuri C6 flow cytometer.

810
S. S. Gehrke et al. / Bioorg. Med. Chem. 21 (2013) 805–813
3H, CH3), 3.30–3.60 (m, 2H, CH2NH), 4.23–4.50 (m, 2H, NCH2),
5. Experimental section
5.1. General
6.60–8.78 (m, 7H, 7 ꢁ ArCH). IR (KBr, cmꢂ1): 2400–3300 (mOH
,
mNH LR-MS: m/z 344 (100%, [M+H]+), 219 (42%), 191 (31%).
5.2.3. 3,4-Dihydroxy-2-methyl-1-{4-[(7-chloro-4-
quinolyl)amino]butyl}pyridiniumchloride (2c)
All chemicals were purchased from commercial sources and
were used as received, unless otherwise noted. Compounds 1a–d
were prepared as previously reported.32 Dry solvents were ob-
tained commercially or dried under standard procedures. Reac-
tions were carried out under normal atmosphere, unless stated
otherwise. Reaction products were purified by flash chromatogra-
phy and characterized by 1H NMR, 13C NMR, ESI-MS, HRMS-MS,
IR and TLC. Flash chromatography columns were packed wet and
the separation was carried out at 1 bar. NMR spectra were recorded
on a Varian VXR 400S spectrometer at 400 MHz or on a PerkinEl-
mer R32 at 90 MHz. Chemical shifts (d) are reported in ppm and
coupling constants (J) in Hz. Signals are referenced to the residual
protons of the respective deuterated solvent. High- and low-reso-
lution mass spectra (LR-MS and HR-MS) were obtained on a LTQ
Orbitrap XL mass spectrometer with electrospray ionization at
the EPSRC National Mass Spectrometry Service Centre, Swansea.
IR spectra were recorded on a PerkinElmer 298. Analytical HPLC
was carried out on a PerkinElmer Series 200 machine equipped
Strategy A: To a solution of 7 (1.3 g, 0.006 mol) in aq ethanol
(83% v/v, 30 mL) was added 3c (1 g, 0.004 mol) followed by aq
NaOH (2 N) until pH 13 was reached. The mixture was refluxed
over night, concentrated and extracted with CHCl3 (3 ꢁ 50 mL).
The combined organic layers were dried over anhydrous sodium
sulfate, filtered and concentrated. Purification by flash chromatog-
raphy (CHCl3/MeOH 8:2 + 0.25–0.3% aq NH3) afforded 0.9 g of 6c as
a yellow-orange solid. 1H NMR (400 MHz, DMSO-d6, ppm) d: 1.61–
1.74 (m, 4 H, b, c-CH2), 2.16 (s, 3H, CH3) 3.38–3.44 (m, 2H, d-CH2),
3
3.94 (t, 2H, JH,H = 6.8 Hz,
a-CH2), 5.00 (s, 2H, CH2), 6.16 (d, 1H,
3JH,H = 7.4 Hz, 3-HPO-CH), 6.65 (d, 1H, JH,H = 6.3 Hz, 3-Ar-CH),
3
3,4
7.29–7.39 (m, 5H, 5 ꢁ Ar-CH), 7.56 (dd, 1H,
J
= 2.2, 9.0 Hz, 6-
H,H
3
Ar-CH), 7.68 (d, 1H, JH,H = 7.4 Hz, 2-HPO-CH), 7.91–7.97 (m, 1H,
3
8-Ar-CH), 8.45 (d, 1H, JH,H = 6.3 Hz, 2-Ar-CH), 8.58 (d, 1H,
3JH,H = 9.0 Hz, 5-Ar-CH), 8.69–8.74 (m, 1H, NH). 13C NMR
(400 MHz, DMSO-d6, ppm) d: 11.9 (CH3), 24.4, 27.7 (b-,
c-CH2),
42.2 (d-CH2), 52.4 ( -CH2), 71.8 (CH2), 98.6 (3-Ar-CH), 116.0 (3-
a
with a Supelcosil™ LC-18-T column (5
l
m, 25 cm ꢁ 4.6 mm), a col-
HPO-CH), 116.5 (Ar-C), 123.1 (8-Ar-CH), 125.3 (5-Ar-CH), 125.4
(6-Ar-CH), 127.8 (Ar-CH), 128.2 (Ar-CH), 128.4 (Ar-CH), 135.7
(Ar-C), 137.7 (Ar-C), 139.6 (2-HPO-CH), 140.8 (Ar-C), 143.7 (Ar-
C), 145.3 (Ar-C), 147.1 (2-Ar-CH), 152.8 (Ar-C), 171.8 (C@O). LR-
MS: m/z 447.1 (100%); HR-MS: 447.0729 [M]ꢂ, calculated for
umn oven (set to 35 °C), and a diode array detector. Compound
purity was determined under the following conditions: 0–100%
MeOH over 60 min (flow rate: 1 mL/min); detection wavelengths:
280 and 354 nm. Thin layer chromatography (TLC) was performed
on pre-coated aluminum plates (Silica Gel 60 F254, Merck). Com-
pounds were visualized by exposure to UV light (254 nm wave-
length) or by staining with ninhydrin, molybdic acid or KMnO4
solution.
C26H26ClN3O2: 447.1714. This material was dissolved in aq HCl
(6 N, 30 mL) and the solution was heated to reflux for 2 h to re-
move the protecting group. The reaction mixture was concentrated
in vacuo, and the resulting precipitate was recrystallized from eth-
anol/acetone, to give the title compound 2c as a white solid. Strat-
egy B: A mixture of 8 (2.7 g, 0.0094 mol) and 5 (1.86 g, 0.0094 mol)
in DMSO (30 mL) was heated to 100–110 °C overnight. The solvent
was removed in vacuo and the residue was purified as described in
Strategy A, to give 2.1 g of 6c as a white solid in 50% yield. Depro-
tection of 6c and subsequent purification was carried out as de-
scribed in Strategy A, to give the title compound 2c as a white
solid. Mp: 130.1–131.9 °C. HPLC: 20.6 min (99%). 1H NMR
5.2. Synthesis
5.2.1. 3,4-Dihydroxy-2-methyl-1-{4-[(7-chloro-4-
quinolyl)amino]ethyl}pyridinium chloride (2a)
To a solution of 7 (1.5 g, 0.0068 mol) in aq ethanol (83% v/v,
36 mL) was added 3a (2.2 g, 0.010 mol). The pH was adjusted to
13 with aq NaOH (10 N) and the reaction was heated to reflux
for 12 h. The solvents were removed and the residue was dissolved
in water. The pH was adjusted to 1 with aq HCl, and the aqueous
solution was washed with ether. The pH was adjusted to 7, and
the aqueous solution was extracted with CHCl3. The combined or-
ganic extracts were dried over NaSO4 and the solvent was removed
in vacuo. To remove the protecting group, the residue was taken up
in aqueous HCl (6 N, 30 mL) and heated to reflux for 2 h. The sol-
vents were removed and the resulting solid was recrystallized from
methanol to give 1.1 g of the title compound as a white solid (39%
yield). Mp: 296–303 °C. 1H NMR (90 MHz, DMSO-d6, ppm) d: 2.42
(s, 3H, CH3), 3.88 (m, 2H, CH2NH2), 4.57 (m, 2H, NHCH2), 6.70–
(400 MHz, DMSO-d6, ppm) d: 1.68–1.79 (m, 2H,
c-CH2), 1.83–
1.92 (m, 2H, b-CH2), 2.53 (s, 3H, CH3), 3.54–3.61 (m, 2H, d-CH2),
3
3
4.38 (t, 2H, JH,H = 7.4 Hz,
a-CH2), 6.89 (d, 1H, JH,H = 7.2 Hz,
3
5-HPO-CH), 7.18 (d, 1H, JH,H = 7.0 Hz, 3-Ar-CH), 7.77 (dd, 1H,
3,4
4
J
= 2.1, 9.1 Hz, 6-Ar-CH), 8.07 (d, 1H, JH,H = 2.1 Hz, 8-Ar-CH),
H,H
3
3
8.26 (d, 1H, JH,H = 7.0 Hz, 2-Ar-CH), 8.55 (d, 1H, JH,H = 7.2 Hz,
3
6-HPO-CH), 8.77 (d, 1H, JH,H = 9.1 Hz, 5-Ar-CH), 9.56 (t(br), 1H,
3JH,H = 5.4 Hz), 14.27 (s(br), 1H, OH). 13C NMR (400 MHz, DMSO-
d6, ppm) d: 12.5 (CH3), 24.2 (CH2), 26.9 (CH2), 42.5 (CH2), 55.3
(CH2), 98.6 (20-HPO-CH), 110.7 (3-Ar-CH), 115.5 (Ar-C), 119.1 (8-
Ar-CH), 125.9 (5-Ar-CH), 126.9 (6-Ar-CH), 138.0 (Ar-C), 138.5 (2-
Ar-CH), 142.9 (Ar-C), 143.2 (Ar-C), 155.4 (Ar-C), 166.3 (C@O). IR
8.40 (m, 7H, 7 ꢁ ArCH). IR (KBr, cmꢂ1): 2400–3300 (mOH
, mNH). LR-
MS: m/z 330 (100%, [M+H]+), 154 (63%).
(KBr, cmꢂ1
[M+H]+), 233 (75%), 154 (38%).
) 2500–3300 (mOH, mNH). LR-MS: m/z 358 (100%,
5.2.2. 3,4-Dihydroxy-2-methyl-1-{4-[(7-chloro-4-
quinolyl)amino]propyl}pyridinium chloride (2b)
5.2.4. 3,4-Dihydroxy-2-methyl-1-{4-[(4-
To a solution of 7 (1.5 g, 0.006 mol) in aq ethanol (83% v/v,
60 mL) was added 3b (2.07 g, 0.010 mol) followed by aq NaOH
(2 N) until pH 12.5 was reached. The reaction mixture was heated
to reflux overnight. The solvents were removed and the residue
was purified by column chromatography (CHCl3:MeOH 9:1 + 0.9%
NH3) to afford 4.3 g of 6b (62% yield). To remove the protecting
group, this material was dissolved in aqueous HCl (12 N, 30 mL)
and heated to reflux for 2 h. Recrystallisation from methanol/ether
gave the title compound as a white solid. Mp: 253.3–254.8 °C. 1H
NMR (90 MHz, DMSO-d6, ppm) d: 1.90–2.40 (m, 2H, CH2), 2.40 (s,
quinolyl)amino]butyl}pyridinium chloride (2d)
The title compound was prepared from 8 and 5 as described in
the synthesis for 9c (Strategy B), but with a different deprotection
procedure. The primary reaction product 6c was dissolved in etha-
nol, the pH was adjusted to 1 with aq HCl, and hydrogenation was
carried out in the presence of 5% Pd/C to give the title compound
2d as a white solid. Mp: 216.5–221.0 °C. 1H NMR (400 MHz, D2O,
ppm) d: 1.80–1.89 (m, 2H, CH2), 1.98–2.06 (m, 2H, CH2), 2.50 (s,
3
3H, CH3), 3.58 (t, 2H, JH,H = 6.6 Hz, CH2), 4.38 (t, 2H, 3JH,H = 6.9 Hz,
3
CH2), 6.65 (d, 1H, JH,H = 7.0 Hz, 3-HPO-Ar-CH), 6.93 (d, 1H,

S. S. Gehrke et al. / Bioorg. Med. Chem. 21 (2013) 805–813
811
3
3JH,H = 6.9 Hz, 3-Ar-CH), 7.65 (at, 1H, JH,H = 7.6, 8.4 Hz, 7-Ar-CH),
5.2.9. 2-Methyl-3-benzyloxy-4-(4H)-pyranone (7)
3
3
7.75 (d, 1H, JH,H = 8.4 Hz, 5-Ar-CH), 7.90 (at, 1H, JH,H = 7.6,
To a solution of maltol (50 g, 0.397 mol) in methanol (450 mL)
was added aq sodium hydroxide (8.7 M, 50 mL of) and benzylbro-
mide (74.66 g, 0.437 mol). The reaction mixture was heated to re-
flux for 6 h. After removal of solvent in vacuo, the oily residue was
taken up in DCM (190 mL), washed with aq sodium hydroxide (5%,
2 ꢁ 190 mL) and water (2 ꢁ 190 mL). The organic fraction was
dried over anhydrous sodium sulfate, filtered and concentrated
to yield as an orange oil which solidified upon cooling. Recrystalli-
zation from Et2O gave 69.9 g of 7 as colorless needles (81% yield).
Mp: 33–34 °C. 1H NMR (90 MHz, DMSO-d6, ppm) d: 1.99 (s, 3H,
CH3), 5.00 (s, 2H, CH2), 6.14–6.24 (m, 1H, ArCH), 7.20 (s, 5H,
5 ꢁ ArCH), 7.39–7.49 (m, 1H, ArCH).
3
8.4 Hz, 6-Ar-CH), 7.99 (d, 1H, JH,H = 6.9 Hz, 2-Ar-CH), 8.03 (d, 1H,
3
3JH,H = 8.4 Hz, 8-Ar-CH), 8.21 (d, 1H, JH,H = 7.0 Hz, 2-HPO-Ar-CH).
13C NMR (400 MHz, D2O, ppm) d: 15.1 (CH3), 26.1 (CH2), 29.3
(CH2), 45.1 (CH2), 59.2 (CH2), 100.8(3-HPO-Ar-CH), 113.6
(3-Ar-CH), 119.4 (Ar-C), 122.8 (5-Ar-CH), 124.7 (8-Ar-CH), 130.0
(7-Ar-CH), 136.6 (6-Ar-CH), 140.1 (Ar-C), 141.5 (2-Ar-CH), 144.2
(2-HPO-Ar-CH), 145.7 (Ar-C), 158.5 (Ar-C), 161.6 (C@O). IR (KBr,
cmꢂ1): 2500–3500 (mOH NH). LR-MS: m/z 324 (45%, [M+H]+), 233
, m
(100%).
5.2.5. 3,4-Dihydroxy-2-methyl-1-{4-[2-methyl-4-
quinolyl)amino]butyl}pyridinium chloride (2e)
A solution of 8 (2.5 g, 0.0087 mol) and 4-chloroquinaldine
(1.5 g, 0.0087 mol) in DMSO was maintained at 110 °C overnight.
The solvent was removed in vacuo and the residue was purified
by column chromatography (CHCl3/MeOH 8:2 + 0.5% NH3) to af-
ford 2.0 g of 6d (54% yield). To remove the protecting group, this
material was dissolved in aqueous HCl (6 N, 50 mL) and heated
to reflux for 2 h. The solvent was removed in vacuo. Recrystalliza-
tion of the residue from methanol/ether afforded the title
compound 2e as a white solid. Mp: 180 °C. 1H NMR (90 MHz,
DMSO-d6, ppm) d: 1.70 (m, 4H, 2 ꢁ CH2), 2.40 (s, 3H, CH3), 2.54
(s, 3H, CH3), 3.30–3.55 (m, 2, CH2NH), 4.10–4.45 (m, 2H, CH2N),
5.2.10. 1-(40-Aminobutyl)-2-methyl-3-benzyloxy-4-(1H)-
pyridone (8)
To a solution of 7 (4.1 g, 0.046 mol) in aq ethanol (50%, 160 mL)
was added 1,4-diaminobutane (10 g, 0.046 mol). The pH of the
reaction mixture was adjusted to pH 13 with aq sodium hydroxide
(10 N). The reaction was heated to reflux for 18 h. The solvents
were removed in vacuo and the residue was taken up in CHCl3.
The organic was washed with water, dried over anhydrous sodium
sulfate, and concentrated in vacuo. The residue was purified by col-
umn chromatography (CHCl3:MeOH 8:2 + 0.5–2% NH3) to afford
6.34 g of 8 as an orange oil (48% yield). 1H NMR (90 MHz, DMSO-
d6, ppm) d: 0.7–1.6 (m, 4H, 2 ꢁ CH2), 1.90 (s, 3H, CH3), 2.13–2.34
(m, 2H, CH2NH2), 2.68 (s, 2H, NH2), 3.41–3.66 (m, 2H, NCH2),
4.70 (s, 2H, CH2Bn), 5.73–5.88 (m, 1H, CHC@O), 7.02 (s, 5H,
5 ꢁ ArCH), 7.19–7.31 (m, 1H, CHCHN).
6.50–8.90 (m, 7H, 7 ꢁ ArCH). IR (KBr, cmꢂ1) 2750–3500 (mOH
,
m
NH). LR-MS: m/z 338 (100%, [M+H]+), 213 (42%).
5.2.6. 7-Chloro-4-(20-aminoethyl)quinoline (3a)
A mixture of 5 (5 g, 0.025 mol) and ethylene diamine (13.485 g,
0.224 mol) was heated to reflux for 12 h. The crude product was
purified by column chromatography (CHCl3:MeOH 9:1 + 4% NH3).
The solvents were removed in vacuo and the brown solid was
washed with CHCl3, to give 3.60 g of 3a as a white solid (66% yield).
Mp: 228.5–230.1 °C. 1H NMR (90 MHz, DMSO-d6, ppm) d: 2.80–
2.97 (m, 2H, CH2-NH2), 3.25–3.47 (m, 2H, NHCH2), 6.15–6.80 (m,
3H, NH, NH2), 7.01–8.24 (m, 5H, 5 ꢁ ArCH).
5.2.11. 3,4-Dihydroxy-2-methyl-1-{4-[(2-methyl-4-
quinolyl)amino]4-oxo-butyl}pyridinium chloride (10)
To a solution of 7 (5 g, 0.023 mol) in ethanol (60 mL) was added
a solution of GABA (2.75 g, 0.276 mol) in water (60 mL). Aq NaOH
(10 N) was added to the reaction until pH 13 was attained. The
reaction mixture was heated to reflux for 18 h. The solvent was re-
duced to 200 mL and the pH was adjusted 4 with aq HCl. The aque-
ous solution was extracted with DCM (3 ꢁ 50 mL). The combined
organic layers were dried over anhydrous sodium sulfate and con-
centrated to afford 4.9 g of 9 as a white solid. This material was
used in the next step without further purification. A mixture of 9
(6 g, 0.020 mol), 4-aminoquinaldine (3.15 g, 0.020 mol) and DCC
(3.55 g, 0.022 mol) in DMF (30 mL) was heated with stirring at
70 °C overnight. The solvent was removed in vacuo and the residue
was purified by column chromatography (CHCl3:MeOH 9:1 + 0.5%
NH3) to afford an orange oil which solidified upon cooling. Recrys-
tallization from MeOH/Et2O gave 5.4 g of benzylated 10 as white
crystals (61% yield). To remove the protecting group, this material
was dissolved in ethanol and hydrogenated in the presence of 5%
Pd/C to give 10 as a white solid. Mp: 275 °C. 1H NMR (90 MHz,
DMSO-d6, ppm) d: 1.69–1.90 (m, 2H, CH2), 1.98 (s, 3H, CH3), 2.48
(m, 2H, CH2), 2.51 (s, 3H, CH3), 3.45–3.75 (m, 2H, CH2N), 4.80 (s,
2H, CH2Bn), 5.90–6.01 (d, 1H, CHC@O), 6.88–7.80 (m, 5H,
5 ꢁ ArCH), 7.00 (s, 5H, 5 ꢁ ArCH), 8.16–8.20 (m, 1H, CH-N). IR
5.2.7. 7-Chloro-4-(30-aminopropyl)quinolone (3b)
A mixture of 5 (5.0 g, 0.025 mol) and 1,3-diaminopropane
(15 mL, 0.176 mol) was heated to reflux for 12 h. The reaction mix-
ture was concentrated and the crude product was washed with
CHCl3. The residue was purified by column chromatography
(CHCl3:MeOH 9:1 + 1% NH3). Subsequent recrystallization from
MeOH/Et2O afforded 4.4 g of 3b as a pale-yellow solid (74% yield).
Mp: 104.5–105.5 °C. 1H NMR (90 MHz, DMSO-d6, ppm) d: 1.50–
1.82 (m, 2H, CH2), 2.41–2.65 (m, 2H, CH2NH2), 3.03–3.30 (m, 2H,
NHCH2), 6.20–8.30 (m, 5H, 5 ꢁ ArCH).
5.2.8. 4-(40-Aminobutylamino)-7-chloro-quinoline (3c)
A mixture of 5 (5 g, 0.025 mol) and 1,4-diaminobutane (11.02 g,
0.125 mol) was heated to reflux for 18 h at 160–180 °C. To the sus-
pension was added chloroform and the mixture was concentrated
in vacuo. Trituration of the resulting solid with DMF followed by
filtration and washing with chloroform yielded a white solid.
Recrystallization from MeOH/Et2O gave 3.85 g of 3c as white crys-
tals (62% yield). Mp: 242.2–244.8 °C. 1H NMR (400 MHz, DMSO-d6,
(KBr, cmꢂ1): 2500–3500 (mOH
352 (100%), 227 (51%), 154 (40%).
, mNH), 1500 (mNH-C@O). LR-MS: m/z
ppm) d: 1.49–1.57 (m, 2H, b-CH2), 1.61–1.70 (m, 2H,
c
-CH2), 3.11–
5.3. Ligand pKa values and stability constant of iron(III)
3.16 (m, 2H, d-CH2), 3.24–3.30 (m, 2H,
a
-CH2), 6.48 (d, 1H,
complexes
3
3JH,H = 5.5 Hz, 3-Ar-CH), 7.37 (t(br), 1H, JH,H = 5.1 Hz, NH), 7.77
4
(d, 1H, JH,H = 2.2 Hz, 8-Ar-CH), 7.97–8.05 (m, 2H, NH2), 8.28 (d,
Iron chloride (17.906 mM in 1% HCl, atomic absorption stan-
dard, Aldrich) was utilized in this study. Analytical grade reagent
HCl (37%) and KOH (10 M) ampoules (Fisher), HPLC grade water
and methanol (Fisher) were used in the preparation of all solutions.
The automatic titration system used in this study comprises of an
autoburette (Metrohm Dosimat 765, l mL syringe) and Mettler
3
3
1H, JH,H = 9.0 Hz, 5-Ar-CH), 8.39 (d, 1H, JH,H = 5.5 Hz, 2-Ar-CH).
13C NMR (400 MHz, DMSO-d6, ppm) d: 25.2 (
-CH2), 26.7 (b-CH2),
36.8 (d-CH2), 42.0 ( -CH2), 98.7 (3-Ar-CH), 117.4 (Ar-C), 124.1 (6-
c
a
Ar-CH), 124.2 (5-Ar-CH), 127.3 (8-Ar-CH), 133.5 (Ar-C), 150.2
(Ar-C), 151.7 (2-Ar-CH), 161.0 (Ar-C).

812
S. S. Gehrke et al. / Bioorg. Med. Chem. 21 (2013) 805–813
Toledo MP230 pH meter with Metrohm AgCl electrode
(6.0133.100) and a reference electrode (6.0733.100). 0.1 M KCl
electrolyte solution was used to maintain the ionic strength. The
temperature of the test solutions was maintained at 25 0.1 °C
by a Techne TE-8J temperature controller. The solution under
investigation was stirred vigorously. A Gilson mini-plus3 pump
with speed capability (20 mL/min) was used to circulate the test
solution into a Hellem quartz flow cuvette.39 For stability constant
determinations, a 50 mm path length cuvette was used and for pKa
determinations a cuvette path length of 10 mm was used. Auto-
matic titration and spectral scans adopted the following protocol:
the pH value of the solution was increased or decreased by 0.1
pH unit by the addition of KOH or HCl from the autoburette over
the pH range of 2–12. When pH readings varied by <0.001 pH unit
over a 3 s period, the solution was allowed to reach equilibrium
(for stability constant determinations a period of 5 min was
adopted, for pKa determinations a period of 1 min was adopted).
The spectrum of the solution was then recorded. The cycle was
then repeated automatically, until the defined end point pH value
was achieved. Following electrode calibration, samples with an
appropriate concentration to give measurable absorbance were ti-
trated in pH range 2–12 for pKa determination. For affinity studies,
the iron:ligand ratio was kept at 5:1 ([iron]total 1 mM). Insoluble ir-
on(III) complexes were measured in 1:1 methanol/water mixtures,
the results were converted to those corresponding to aqueous con-
ditions. The titration data were analyzed by pHab.39,40 Affinity is
expressed as the pM value. pM is defined as the negative logarithm
of the metal ion concentration under the following conditions:
[Metal Ion]total = 10ꢂ6 M, [Ligand]total = 10ꢂ5 M at pH 7.4.
of 100
l
M for 24 h at 37 °C in a 5% CO2-humidified incubator. Ben-
znidazole was used as a positive control (GI50 440.7
lM, CI95%
406.2–478.4). The trypomastigote viability was based on the
cellular conversion of the soluble tetrazolium salt MTT
(3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyl-tetrazolium bromide)
into the insoluble formazan by mitochondrial enzymes.34,35
5.4.4. Anti-leishmanial activity assay
The anti-leishmanial activity of compounds was determined
against L. infantum (MHOM/BR/1972/LD) promastigotes. Parasites
were maintained in M-199 medium supplemented with 10% calf
serum and 0.25% hemin at 24 °C. Compounds were incubated to
the highest concentration of 100 lM for 48 h at 24 °C and the via-
bility of parasites was colorimetrically determined by the mito-
chondrial oxidation of MTT. Amphotericin B was used as a
positive control (GI50 0.17 lM, CI95% 0.14–0.21). Compounds were
also tested against intracellular amastigotes of L. infantum, using
peritoneal macrophages as host cells. Compounds were incubated
for 120 h at 37 °C in a 5% CO2-humidified incubator, using meglu-
mine antimonate (Glucantime) as a standard drug. The infection
rate was determined by light microscopy by the number of in-
fected macrophages out of 500 cells in duplicate.42
Acknowledgement
We thank the Norwich Research Park for a studentship (to
S.S.G.), and King’s College London (King’s Brazil Initiative) and FA-
PESP Sao Paulo (project 2011/50577–7) for a pump prime award
(to G.K.W. and A.T.).
5.4. Biological assays
Supplementary data
The activity of test compounds against P. falciparum (strain
ITG2G1) was determined as previously described.41 Other bioas-
says were carried out as follows:
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.009.
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